A peroxidase is an enzyme, which may contain heme, that catalyzes a reaction of the form:ROOR′+electron donor (2e′)+2H+→ROH+R′OH
For many of these enzymes the optimal ROOR′ (electron acceptor) substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides.
The nature of the electron donor is very dependent on the structure of the enzyme. For example, horseradish peroxidase (HRP), which is isolated from horseradish (Armoracia rusticana) roots, can use a variety of organic compounds as electron donors and acceptors.
Being a member of the ferroprotoporphyrin group of peroxidases, HRP does contain a heme group. Located at positions distal and proximal to the heme plane there are in addition two calcium binding sites. HRP C dominates quantitatively among the isoperoxidases of horseradish root. HRP C is a single chain polypeptide comprising 308 amino acid residues which form four internal disulfide bridges and 8 neutral carbohydrate side-chains. The molecular weight of the polypeptide chain is 33890 Daltons (Da), and the molecular weight of native horseradish peroxidase C is about 44 kDa (Welinder, K. G., Eur. J. Biochem. 96 (1979) 483-502). At least seven isozymes of HRP exist (Shannon, L. M. et al., J. Biol. Chem 241 (1966) 2166-2172). The carbohydrate composition consists of galactose, arabinose, xylose, fucose, mannose, amnnosamine, and galactosamine, depending upon the specific isozyme (Shannon, L. M., et al. J. Biol. Chem. 241 (1966) 2166-2172). The isoelectric point of the isozymes ranges from 3.0 to 9.0. The pH optimum of HRP is in the range of pH 6.0 to pH 6.5; activity at pH 7.5 is 84% of the maximum. The enzyme is most stable in the range of pH 5.0 to pH 9.0 (Schomburg, D., et al., Enzyme Handbook 7 (1994) EC 1.11.1.7:1-6).
HRP combines with hydrogen peroxide (H2O2) and the resultant [HRP-H2O2] complex can oxidize a wide variety of chromogenic hydrogen donors. HRP has a broad and accessible active site and many chemically very different compounds can reach the site of the reaction. Known HRP substrates include TMB (3,3′,5,5′-tetramethylbenzidine), ABTS (2,2′-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid diammonium salt), luminol (5-amino-1,2,3,4-tetrahydrophthalazin-1,4-dion) and isoluminol (4-aminophthalhydrazide), as well as fluorogenic substrates such as tyramine (4-hydroxy-phethylamine), homovanillic acid, and 4-hydroxyphenyl acetic acid. Further HRP substrates are known to the art. Due to its versatility, HRP is commercially used as a component of immunoassays, such as coupled enzyme assays, chemiluminescent assays and assay kits for clinical diagnostics including histochemistry kits.
In a typical example, an immunoassay based on a sandwich ELISA principle and using analyte-specific coating and capture antibodies includes HRP conjugated to the capture antibody. The peroxidase enzyme catalyzes the cleavage of a chromogenic substrate to yield a product which can be measured spectrophotometrically. The absorbance of a colored or fluorescent product is directly correlated to the amount of analyte in the sample analyzed. In order to allow the comparison of simultaneous measurements including controls, the HRP enzymatic reaction needs to be terminated after a defined incubation period. To this end a stop reagent is used. Stop reagents have to fulfill two major requirements: (1) to terminate the reaction by effectively inhibiting the enzymatic activity of HRP; (2) to stabilize the oxidized products of the chromogenic or fluorogenic substrate(s).
In Zollner, H., Handbook of Enzyme Inhibitors, 2nd Ed. (1989) Part A: 367-368) the following compounds have been described as inhibitors of HRP: sodium azide, cyanide, L-cystine, dichromate, ethylenethiourea, hydroxylamine, sulfide, vanadate, p-aminobenzoic acid, Cd+2, Co+2, Cu+2, Fe+3, Mn+2, Ni+2, Pb+3. Many stop reagents known to the art make use of these compounds. Another known reagent used to stop the HRP activity is oxalic acid.
When designing a stop reagent for reactions catalyzed by HRP, bleaching of the color brought about by the chromogenic substance is a frequent problem. The use of heavy metal salts in stop reagents has a number of disadvantages including toxicity. Also, certain salts of heavy metals are explosive as dry materials. Another known stop reagent for the HRP reaction is formaldehyde. However, the stabilizing effect of this compound is unsatisfactory. In addition, formaldehyde is toxic and has a troublesome smell.
U.S. Pat. No. 4,234,680 teaches the use of an alkali metal bisulphite as stop reagent. However, this reducing agent has the potential of decolorizing the oxidized products formed from certain chromogenic substrates by the HRP catalyzed reaction. This applies especially to the oxidized products of ABTS and related salts.
In U.S. Pat. No. 4,752,570 a process for the determination of peroxidase is described, wherein a chromogenic substrate reaction is stopped by adding catalase as stop reagent. This process, however, requires the use of an enzyme with limited stability and causes foaming due to the release of oxygen. Foaming may interfere with spectrophotometric readings.
Finally, surface-active agents, such as secondary alkyl sulphate or dodecyl hydrogen sulphate, have been suggested as stop agents. For example, SDS (sodium dodecyl sulphate) at a final concentration in solution of 0.5% [w/v] is suggested for stopping the color formation using HRP and ABTS, see pack insert of product #11684302 (catalogue of Roche Diagnostics GmbH, Mannheim, Germany). Similarly, an SDS solution is supplied from KPL laboratories for the same purpose. However, these compounds are not able to fully suppress further color formation and/or lead to precipitation of the substrate. Results presented in Example 1 illustrates this fact.
Also strong (mineral) acids are used as stop reagents to inhibit peroxidase enzymatic activity. This is especially the case when 3,3′-5,5′-tetramethylbenzidine serves as a color substrate and the peroxidase enzyme is inhibited by adjusting the pH to values of pH 2 or even lower.
Several reagents that are described above lead to precipitation of colored substrates like ABTS and cause erroneous readings, e.g. spectrophotometric readings of ELISA plates. Precipitation particularly occurs when using strong acids at pH values lower than 2. In addition, 1 M H2So4 changes the color of the substrate 3,3′-5,5′-tetramethylbenzidine from blue to yellow. Other inhibitors (e.g. bisulphate, see above) may lead to bleaching and therefore are of limited use. Furthermore, certain stop reagents only after a certain lag phase achieve inhibition or just lead to an incomplete inhibition of the HRP-catalyzed reaction.
It is an object of the present invention to overcome the disadvantages of the stop reagents of the state of the art. It is a particular object of the present invention to provide an improved stop reagent for HRP-catalyzed reactions. Another object of the invention is to provide a process for the determination of peroxidase enzymatic activity in a sample.